The invention relates generally to novel methods of treating mammalian diseases using compounds that inhibit a biological activity of a topoisomerase enzymes.
DNA topoisomerases are a group of enzymes present in all cells (both prokaryote and eukaryote) which are responsible for catalyzing topological changes in DNA. These enzymes have important functions for DNA replication, transcription and recombination, and have been shown to be essential for viability. Briefly, DNA topoisomerases supercoil and relax polynucleotides; they catalyze the reaction in which a polynucleotide, such as a double stranded DNA, wraps around the enzyme forming a complex therewith. The enzyme then catalyzes the cleavage of the double-stranded DNA and the passage of another DNA segment through the cleavage site, and then the relegation of the DNA at the cleavage site.
Eukaryotic topoisomerases are targets for antitumor agents. Some chemical agents, e.g., certain poisons and catalytic inhibitors, able to interfere with DNA topoisomerases have clinical efficacy as antitumor drugs. Many of these agents inhibit the topoisomerase at different sites, resulting in differential anticancer activity. [G. Capranico and M. Binaschi, (1998) Biochim et Biophs. Acta, 1400:185-1941. In particular, the quinolone group and the coumarin antibiotics, e.g., novobiocin and coumermycine A1 [A. Maxwell, (1997) xe2x80x9cDNA Gyrase as a Drug Targetxe2x80x9d, Trends in Microbiology, 5:102-109] are useful anti-bacterial drugs. DNA cleavage is not required for the binding of quinolone drugs to the gyrase-DNA complex [Critchlow S. E., and Maxwell A. (1996) Biochemistry, 35: 7387-7393], the antibacterial activity of the quinolone group of antibacterials is, however, predicated upon the ability of these compounds to induce gyrase-mediated DNA breakage. [Drlica, K. and Zhao, X. (1997) Microbiology and Molecular Biology Reviews, 61:377-392]. See, also, M. D""Incalci, (1993) Curr. Opin. Oncol., 5:1023-1028; Y. Pommier, (1988) Biochimie, 80:255-270; A. Maxwell, (1996) Nature Structural Biol., 3(2):109-112; G. Capranico et al, Chap. 6 in xe2x80x9cCancer Chemotherapy and Biological Response Modifiers Annual 17xe2x80x9d, H. M. Pinedo et al eds., 1997, Elsevier Science B. V.; and M. Couteurier et al, (1998) Trends in Microbiology, 6(7):269-275; Osheroff, J. Biol. Chem., 261:9944-9950 (1985); and Osheroff, Pharinac. Ther., 41:223-241 (1989)].
Among the inhibitors of human topoisomerase II are merbarone, and the bis(2,6-dioxopiperazines) such as ICR-193. While these agents do not induce topoisomerase dependent cleavage of DNA, their mechanism of action is different from the subject of this invention. Merbarone inhibits the catalytic activity of human topoisomerase 11 by blocking DNA cleavage, however DNA binding studies showed that the apparent Kd""s (dissosciation constants) for enzyme to DNA binding were not substantially affected by the inhibitor, thus not consistent with the stabilisation of a ternary complex (Fortune J. M., and Osheroff N. (1998) J. Biol. Chem. 273 17643-50). ICRF-193 blocks ATP hydrolysis in eucaryotic topoisomerase II, an action that traps the enzyme on the DNA in a closed clamp form, preventing the protein clamp from opening, and thereby preventing release of DNA (Roca et al PNAS 91 1781-5). Point mutations, causing resistance to these drugs, however map to the ATPase of topoisomerase II (Wessel et al 1999 Cancer Research 59 3442-50). Additionally, ICRF-193 has been shown to directly bind to the dimerized ATPase domains of the yeast enzyme (Olland S., and Wang J. C. 1999 J. Biol Chem 274 21688-94).
Some of the presently used compounds for antitumor treatment based on inhibition of topoisomerases have disadvantages, such as ineffectiveness against certain cell types due to resistance mechanisms associated with their mode of action, unwanted toxicity and mutagenicity, particularly in view of the DNA cleavage activity of these compounds. Thus, there exists a need in the art for novel anti-tumor compounds, pharmaceutical compositions and methods of use thereof, especially compounds that do not exhibit DNA cleavage activity. Such compounds, compositions and methods are provided by the present invention.
In one aspect, the invention provides a method of modulating the activity of a type II topoisomerase enzyme of an aberrant cell comprising contacting the aberrant cell enzyme with a compound that inhibits the enzyme-mediated cleavage of a polynucleotide substrate. In one embodiment of this method, the compound forms a stable or transient non-covalent complex, preferably a ternary complex, with a topoisomerase enzyme and a substrate, for example a polynucleotide, particularly DNA. In another embodiment of this method, the compound inhibits the formation of the complex between a substrate and the enzyme. The aberrant cell which employs this enzyme for replication may be from a eukaryote, particularly a mammal, and especially a human. The polynucleotide substrate may be any DNA, RNA or DNA-RNA hybrid. In one embodiment, the method involves contacting an enzyme, or an aberrant cell, with the compound that inhibits replication of, or kills, the aberrant cell carrying the enzyme. Such contacting step can occur in vitro, in vivo in a mammal containing the aberrant cell or ex vivo in mammalian tissue outside of the body.
In another aspect, the invention provides a pharmaceutical composition comprising a compound that inhibits, arrests, or otherwise alters, the aberrant cell type II topoisomerase enzyme-mcdiated cleavage of a polynucleotide substrate in a pharmaceutically or physiologically acceptable carrier. In one embodiment, the compound is one that is identified by the assays described herein. Preferably, the composition has anti-tumor, and may contain other agents and/or excipients useful in the treatment of aberrant cell diseases, particularly in mammals, and especially in humans.
In yet a further aspect, the invention provides a method for treating a mammal or mammalian tissue comprising aberrant cells having a type II topoisomerase enzyme, the method comprising administering to the mammal an effective amount of an above-described pharmaceutical composition. This method involves administering the composition by a route, such as intravenous, oral, intradermal, transdermal, intraperitoneal, intramuscular, subcutaneous, by inhalation and mucosal. Preferably this method is useful for treating such diseases in a human, or in human tissue.
In another aspect, the invention provides a method for identifying an anti-tumor compound comprising screening the compound for the ability to inhibit, or otherwise alter, an aberrant cell type II topoisomerase-mediated cleavage of a polynucleotide substrate. In one embodiment, the method includes determining that a compound forms a high molecular weight higher order complex, such as a ternary complex, with the enzyme and a polynucleotide substrate.
In a preferred embodiment a method is provided for identifying an anti-tumor compound comprising screening the compound for the ability to inhibit, or otherwise alter, an aberrant cell type II topoisomerase-mediated cleavage of a polynucleotide substrate in the present of another compound. In this method compounds that potentiate the antagonism of the aberrant cell type II topoisomerase-mediated cleavage may be identified.
In another embodiment a compound may be screened against both a mammalian topoisomerase and a pathogen topoisomerase or gyrase, such as prokaryotic topoisomerase or gyrase, to identify a compound that inhibits and/or binds to both the mammalian topoisomerase and the pathogen topoisomerase or gyrase.
In another aspect of the invention are compounds of the invention not known in the art prior to the filing date of this application or an application to which this application claims benefit of priority.
In one embodiment, the method includes determining that a compound forms a high molecular weight higher order complex, such as a ternary complex, with the enzyme and a polynucleotide substrate.
In another embodiment, the determining step comprises adding a reaction mixture comprising in a buffer, a test compound, the enzyme, and the polynucleotide substrate to a size exclusion chromatographic column; and monitoring the fractions eluting from the chromatographic column to detect the fraction containing the higher order complex, such as a ternary complex.
In another embodiment, the screening method involves detecting an intact complex comprising the polynucleotide and the enzyme. Such a screening method involves reacting a test compound with the enzyme and polynucleotide substrate; quenching the reaction with a quenching compound, such as a denaturant; and performing a detection analysis, such as a gel analysis, to detect if the polynucleotide is intact or altered.
In still another embodiment, the screening method involves performing a replication blockage assay.
In a further aspect, the invention provides a compound identified by any of the above screening methods.
In yet a further preferred embodiment the compound comprises a moeity that binds both subunits of a topoisomerase, or which compound comprises a moiety that binds more than one topoisomerase homo- or hetero-dimers, or which compound binds more than one topoisomerase homo- or hetero-dimer.
In still an additional embodiment, the invention provides a method for modifying a surface comprising contacting a surface with a composition comprising a compound which inhibits an aberrant cell type II topoisomerase-mediated cleavage of a polynucleotide substrate. The surface may be a biological tissue, in or outside of an individual. The method""s contacting step comprises administering a suitable modifying dosage of the composition by means selected from the group consisting of coating, spraying, implanting, or soaking, among others.
In one aspect, the invention provides a method of modulating the activity of a mammalian type II topoisomerase enzyme comprising contacting the enzyme with a compound that inhibits enzyme-mediated cleavage of a polynucleotide substrate. In one embodiment, this method permits the compound to form a transient or stable non-covalent higher order structure, such as a ternary complex, comprising the enzyme, the polynucleotide, and the compound. In another embodiment, the method involves preventing the formation of the enzyme-polynucleotide complex, or comprising the enzyme and the compound. In another embodiment, the method involves preventing the formation of the enzyme-polynucleotide complex. The mammalian enzymes are preferably human or domestic animal in origin. The polynucleotide substrate is a polynucleotide, such as, DNA, RNA or a DNA-RNA hybrid, including but not limited to polynucleotides with modified bases. In a preferred embodiment, the enzyme is associated with a mammalian disease, and the method inhibits the progression of the disease, e.g., cancer. Preferably the method inhibits replication, proliferation or differentiation of cancer cells. The contacting step of the method can occur in vitro, in vivo in a mammal, or ex vivo on mammalian tissue.
In another aspect, the method provides a pharmaceutical composition comprising a compound that inhibits the mammalian type II topoisomerase enzyme-mediated cleavage of a polynucleotide substrate in a pharmaceutically or physiologically acceptable carrier. In one embodiment, the compound is a compound described herein. In another embodiment, the compound is one identified by the screening assays described herein. The composition preferably has anti-cancer activity, and can contain other conventional anticancer agents or excipients normally useful in anticancer compositions.
In still another aspect, the invention provides a method for treating a disease, e.g., cancer, in a mammal characterized by the abnormal behavior of a mammalian type II topoisomerase enzyme comprising administering to the mammal having the disease an effective amount of a pharmaceutical composition described above. According to the method, the composition is administered by a route, such as intravenous, oral, intradermal, transdermal, intraperitoneal, intramuscular, subcutaneous, by inhalation and mucosal in a dosage appropriate for the disease, patient, e.g., human, and route of administration.
In yet another aspect, the invention provides a method for identifying a compound useful to treat mammalian diseases characterized by the aberrant presence or activity of a mammalian type II topoisomerase comprising screening the compound for the ability to inhibit a mammalian type II topoisomerase-mediated cleavage of a polynucleotide substrate. Preferably the compound is an anticancer compound. One method step involves determining that the compound forms a high molecular weight ternary complex with the enzyme and the polynucleotide substrate. In one embodiment such a determining step comprises adding a reaction mixture comprising in a buffer, a test compound, the enzyme, and the polynucleotide substrate to a size exclusion chromatographic column; and monitoring the fractions eluting from the chromatographic column to detect the fraction containing the ternary complex.
In another embodiment of a screening method, a step is performed to detect an intact complex comprising the polynucleotide and the enzyme. For example, a test compound is reacted with the enzyme and polynucleotide substrate; the reaction quenched with a denaturant; and a gel analysis performed to indicate if the polynucleotide is intact. In still another embodiment of a screening method, a screening step comprises a replication blockage assay.
In still another aspect, the invention provides a method for screening for an anticancer compound comprising the steps of: obtaining the crystal structure of a compound that inhibits the mammalian type II topoisomerase-mediated cleavage of a polynucleotide substrate; and performing computer analysis to design or select from among test compounds, a compound having a substantially similar binding characteristics.
In one embodiment, the method comprises the step of exposing the compound having the substantially sinmilar crystal structure to a sample of cancer cells, and observing the cells for inhibition of replication, wherein the occurrence of inhibition is indicative of an anticancer compound.
In yet a further embodiment of the invention, a method of treatment is provide comprising the step of contacting the patient to be treated with a composition comprising compound of the invention and another antineoplastic agent, preferably an antineoplastic agent that acts by a mechanism other than topoisomerase.
Another embodiment of the invention provides a composition comprising compound of the invention and another antineoplastic agent, preferably an antineoplastic agent that acts by a mechanism other than topoisomerase.
In yet a further aspect, the invention provides a compound identified by the methods described above.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.